Accumulation Of Tricarboxylic Acid Cycle Intermediates Research Articles

368-OR: Activation of Hepatic Gluconeogenesis Is Required to Suppress DNL and Stimulate Ketogenesis during Fasting

Carbohydrates and lipids are primary energy sources in mammals; thus, their oxidation and synthesis are carefully controlled. The tricarboxylic acid (TCA) cycle is necessary to oxidize these substrates, and in liver it also supports their synthesis. However, its role in regulating these processes, particularly de novo lipogenesis (DNL) is underappreciated. We report that activation of hepatic gluconeogenesis (GNG) is required to trigger the canonical lipid and carbohydrate response to fasting in mice. Phosphoenolpyruvate carboxykinase (PEPCK) catalyzes cataplerotic flux from the TCA cycle and is required for GNG from lactate, pyruvate and amino acids. Knockdown (KD) or knockout (KO) of PEPCK caused the massive accumulation of TCA cycle intermediates and KO mice developed fatty liver after an overnight fast. Lipidomic profiling of liver suggested increased lipid desaturation and elongation with loss of PEPCK. 2H NMR analysis of liver lipids following 2H2O administration demonstrated elevated fasting DNL in KO mice, which was similar to fed WT mice. In addition, fasting ketogenesis, estimated by infusions of [U-13C]BHB and [3,4-13C]AcAc, was decreased in KO mice. Despite an impaired fasting response, the expression of genes related to control of fat oxidation and DNL were not decreased, suggesting that loss of regulation was mediated by a metabolic mechanism. Inhibition of GNG produced a reduced mitochondrial redox state which limited oxidative pathways and decreased acetyl-CoA concentration. Inhibition of GNG also decreased fasting ATP requirements, resulting in a high energy charge which suppressed activation of AMPK, its canonical suppression of acetyl-CoA carboxylase and caused increased malonyl-CoA during fasting. Hence, PEPCK mediated cataplerosis and GNG during fasting is not only an important source of glucose, it is also necessary to shift cellular redox and energetics to states necessary to suppress lipogenesis and maintain ketogenesis during fasting. Disclosure S. Deja: None. J. Duarte: None. J.A. Fletcher: None. B. Kucejova: None. X. Fu: None. G. Vale: None. J. Young: Board Member; Self; Metalytics. Consultant; Self; Pfizer Inc. J. Browning: None. S.C. Burgess: None. Funding National Institutes of Health (R01DK078184, P41EB015908); Robert A. Welch Foundation (I-1804)

Carbon balance and source-sink metabolic changes in winter wheat exposed to high night-time temperature.

Carbon loss under high night-time temperature (HNT) leads to significant reduction in wheat yield. Growth chamber studies were carried out using six winter wheat genotypes, to unravel postheading HNT (23°C)-induced alterations in carbon balance, source-sink metabolic changes, yield, and yield-related traits compared with control (15°C) conditions. Four of the six tested genotypes recorded a significant increase in night respiration after 4days of HNT exposure, with all the cultivars regulating carbon loss and demonstrating different degree of acclimation to extended HNT exposure. Metabolite profiling indicated carbohydrate metabolism in spikes and activation of the TriCarboxylic Acid (TCA) cycle in leaves as important pathways operating under HNT exposure. A significant increase in sugars, sugar-alcohols, and phosphate in spikes of the tolerant genotype (Tascosa) indicated osmolytes and membrane protective mechanisms acting against HNT damage. Enhanced night respiration under HNT resulted in higher accumulation of TCA cycle intermediates like isocitrate and fumarate in leaves of the susceptible genotype (TX86A5606). Lower grain number due to lesser productive spikes and reduced grain weight due to shorter grain-filling duration determined HNT-induced yield loss in winter wheat. Traits and mechanisms identified will help catalyze the development of physiological and metabolic markers for breeding HNT-tolerant wheat.

Mll-Partial Tandem Duplication (Mll-PTD) Causes Pseudohypoxia and Hypermethylation of the Epigenome through Suppression of Mitochondrial Complex II

The MLL-partial tandem duplication (MLL-PTD),characterized by the internal duplication of exons 3-9 or 3-11 in the MLL gene, produces an elongated protein, and is considered as a gain-of-function mutation. The MLL-PTD is primarily found in elderly patients with myelodysplastic syndromes and acute myeloid leukemiaas well as healthy individuals.Previously we showed that Mll-PTD knock-in (MllPTD/WT) mice presented enhanced self-renewal of hematopoietic stem cells (HSCs) and partially blocked differentiation of hematopoietic stem/progenitor cells (HSPCs). Interestingly, Mll-PTD increased the protein level of HIF1A in HSPCs, which is critical for enhanced self-renewal of HSCs. In the current study, we investigated the mechanisms for HIF1A activation by Mll-PTD.In normoxia, HIF1A is hydroxylated by prolyl hydroxylases (PHD), resulting in rapid protein degradation via ubiquitination. PHD is one of the well-known enzymes whose activity is dependent on the cellular level of α-ketoglutarate (α-KG), one of the metabolites in the tricarboxylic acid (TCA) cycle. Accumulation of subsequent metabolites of α-KG, such as succinate, fumarate, and malate, inhibits activity of α-KG-dependent enzymes. Indeed, mitochondrial dysfunction is known to result in accumulation of TCA cycle intermediates, leading to activation of HIF signaling. Thus, we first examined if Mll-PTD induces the alteration of mitochondrial functions. Interestingly, cellular respiration and activity of mitochondrial complexes (I, II, and III) were significantly decreased in HSPCs of MllPTD/WT mice, while the copy number of mitochondrial DNA was not altered. These results indicate that suppression of mitochondrial activity is not due to the decrease of the total mitochondria. We also examined mRNA expression levels of several major TCA cycle enzymes, and found that succinate dehydrogenase (Sdh) complex (Sdha, Sdhb, and Sdhd) was significantly downregulated in MllPTD/WT HSPCs. SDH is a critical TCA cycle enzyme which converts succinate to fumarate. Inactivation of SDH is known to result in impairment of mitochondrial biogenesis, a blockade of the TCA cycle, and accumulation of TCA cycle metabolites.We next quantified metabolites in glycolysis and TCA cycle in the plasma from WT control and MllPTD/WT mice. NMR analysis revealed that succinate, fumarate, and malate were increased in the plasma of MllPTD/WT mice. Especially, the ratios of fumarate and malate to α-KG were both significantly increased in MllPTD/WT compared to WT control. Indeed, post-α-KG metabolites increased HIF1A protein in human cord blood CD34+cells in vitro, indicating that higher levels of succinate, fumarate, and malate to α-KG levels stabilize HIF1A. We also confirmed that knockdown of Sdh increased the HIF1A protein level in murine cell line in normoxia. These results indicate that downregulation of Sdh in MllPTD/WT is one of the mechanisms for suppression of mitochondrial activity, leading to pseudohypoxia and HIF1A activation.Besides PHD, TET and histone lysine demethylases are also α-KG-dependent enzymes. We found that in MllPTD/WT HSPCs, the 5-methylcitosine (5-mC) level was increased in genomic DNA, and trimethylation levels at H3K4, H3K9, H3K36 and H3K79 were also increased. Collectively, these results suggest that metabolic pseudohypoxia due to lower mitochondrial activity not only activates HIF1A signaling but also induces hypermethylation in DNA and histones, through suppression of α-KG-dependent PHD and demethylases.In summary, we demonstrate that through suppression of mitochondrial complex II, Mll-PTD causes pseudohypoxia and hypermethylation of the epigenome, which may contribute to expansion of premalignant clones and accumulation of additional mutations in those cells. Interestingly, it has been proposed that IDH mutations are involved in tumorigenesis in leukemias and brain tumors through a similar mechanism. Moreover, loss-of-function mutations of the TCA cycle enzymes, SDH complex, and fumarate hydratase, are frequently found in various solid tumors associated with pseudohypoxia and hypermethylation phenotypes. Further investigations of the impact of metabolic-rewiring-mediated pseudohypoxia/hypermethylation on tumorigenesis may lead to the development of novel therapeutic strategies to prevent the onset and/or the progression of various types of malignant diseases. DisclosuresNo relevant conflicts of interest to declare.

The nature of unmeasured anions in critically ill patients

We read with interest the commentary of Venkatesh and Morgan in the February issue of Critical Care [1], in which they discuss the continuing need for clarification of the nature of unmeasured anions in critically ill patients. The questions asked in this commentary arise from the study of Bruegger and colleagues in a previous issue of this journal [2]. The main issue discussed is the ongoing uncertainty about the contribution of intermediates of the tricarboxylic acid (TCA) cycle to the strong ion gap (SIG). As Venkatesh and Morgan accurately point out, the extremely high concentrations of acetate and citrate found by Bruegger and colleagues may very well be confounded by the exogenous administration of resuscitation fluids and blood products in the animal model described. In the March issue of Critical Care Medicine, we published our study conducted in critically ill patients with a metabolic acidosis with and without an increased SIG, representing the absence or presence of unmeasured anions [3]. We explored the presence of and contribution to the SIG of a large variety of compounds, largely organic acids and amino acids, by use of several laboratory techniques such as ion-exchange column chromatography and gas chromatography. In addition, proton nuclear magnetic resonance (1H-NMR) spectroscopy was used to quantify intermediates of the TCA cycle. Despite our elaborate attempt, only 7.9% of the difference in SIG between the low and high SIG groups could be explained by the presence of high concentrations of amino acids, uric acid, and organic acids. As a single compound, uric acid was responsible for the largest relative contribution to the SIG, namely 2.2%. Our study excludes many other potent unmeasured anions, such as uremic toxins and pyroglutamic acid, as major contributors to the SIG. As to the presence of TCA cycle intermediates, only succinic acid was significantly elevated in the high SIG group but it accounted for only 0.07% of the increased SIG. Furthermore, concentrations of isocitrate and citrate obtained by gas chromatography were not relevantly elevated in the high SIG group and all intermediates of the TCA cycle quantified with 1H-NMR spectroscopy, including acetic acid, corresponded to the published reference ranges. As outlined in our discussion, these findings are in contradiction to a recent human study [4]. The accumulation of TCA cycle intermediates by the mechanism of accelerated amino acid catabolism would imply distinct patterns of altered amino acid concentrations, which also was not supported by the results of our study. Our research enables us to answer the last question posed in the commentary of Venkatesh and Morgan. Raised concentrations of (iso)citrate and acetate do not clarify the SIG in the critically ill. Thus, a significant contribution of TCA cycle intermediates to the SIG in human acidosis is excluded. However, given the small percentage of the SIG that was represented by the measured anions, a largely unclarified portion of the strong anion gap remains to be explored.

Impaired Tricarboxylic Acid Cycle Activity in Mouse Livers Lacking Cytosolic Phosphoenolpyruvate Carboxykinase

Liver-specific phosphoenolpyruvate carboxykinase (PEPCK) null mice, when fasted, maintain normal whole body glucose kinetics but develop dramatic hepatic steatosis. To identify the abnormalities of hepatic energy generation that lead to steatosis during fasting, we studied metabolic fluxes in livers lacking hepatic cytosolic PEPCK by NMR using 2H and 13C tracers. After a 4-h fast, glucose production from glycogenolysis and conversion of glycerol to glucose remains normal, whereas gluconeogenesis from tricarboxylic acid (TCA) cycle intermediates was nearly absent. Upon an extended 24-h fast, livers that lack PEPCK exhibit both 2-fold lower glucose production and oxygen consumption, compared with the controls, with all glucose production being derived only from glycerol. The mitochondrial reduction-oxidation (red-ox) state, as indicated by the NADH/NAD+ ratio, is 5-fold higher, and hepatic TCA cycle intermediate concentrations are dramatically increased in the PEPCK null livers. Consistent with this, flux through the TCA cycle and pyruvate cycling pathways is 10- and 40-fold lower, respectively. Disruption of hepatic cataplerosis due to loss of PEPCK leads to the accumulation of TCA cycle intermediates and a nearly complete blockage of gluconeogenesis from amino acids and lactate (an energy demanding process) but intact gluconeogenesis from glycerol (which contributes to net NADH production). Inhibition of the TCA cycle and fatty acid oxidation due to increased TCA cycle intermediate concentrations and reduced mitochondrial red-ox state lead to the development of steatosis.

Role of NADP+ (corrected)-linked malic enzymes as regulators of the pool size of tricarboxylic acid-cycle intermediates in the perfused rat heart.

Cytosolic and mitochondrial concentrations of malate, 2-oxoglutarate, isocitrate and pyruvate in the isolated perfused rat heart were measured by non-aqueous tissue fractionation, taking the NADP-linked isocitrate dehydrogenase as indicator reactions for the free [NADPH]/[NADP+] ratios. The mass-action ratios of NADP-linked malic enzymes (EC 1.1.1.40) were found to be on the side of pyruvate carboxylation by more than one order of magnitude in both the cytosolic and the mitochondrial spaces in hearts perfused with glucose, whereas during propionate perfusion this ratio approached the equilibrium constant (Keq.) of malic enzyme. The results consequently indicate that the NADP-linked malic enzymes cannot be responsible for the feed-out (cataplerotic) reactions from the tricarboxylic acid cycle which occur during glucose perfusion. Only when other anaplerotic fluxes into the cycle are high, as during propionate oxidation, which results in accumulation of tricarboxylic acid-cycle intermediates, is a steady state reached which allows efflux via the malic enzyme.